Simultaneous quadruple-label fluorometric immunoassay of thyroid- stimulating hormone, 17α-hydroxyprogesterone, immunoreactive trypsin, and creatine kinase MM isoenzyme in dried blood spots

Abstract

We describe a quadruple-label fluorometric immunoassay for simultaneously measuring four analytes: thyroid-stimulating hormone (TSH), 17α- hydroxyprogesterone (17α-OHP), immunoreactive trypsin (IRT), and creatine kinase MM (CK-MM). The assay is based on immunoreagents labeled with four different lanthanide ions (Eu3+, Tb3+, Sm3+, and Dy3+), on dissociative fluorescence enhancement applying the principle of co- fluorescence, and on time-resolved fluorometry. The monoclonal anti-α-TSH and anti-IRT antibodies and the polyclonal anti-CK-MM antibody were labeled with Eu3+, Sm3+, and Dy3+, respectively; 17α-OHP was labeled with Tb3+. The assay was performed in microtitration strip wells coated with a mixture of monoclonal antibodies against β-TSH, IRT, and CK-MM and a polyclonal goat anti-rabbit IgG for capture of the rabbit anti-17α-OHP antibodies. After completion of the immunoreactions, the bound fractions of the lanthanides were dissociated into the co-fluorescence enhancement solution, creating highly fluorescent chelates. The four lanthanide-specific signals were subsequently measured in a time-resolved fluorometer. The detection limits of the assay were 0.1 mlU/L for TSH, 2 nmol/L for 17α-OHP, 2 μg/L for IRT, and 4 U/L for CK-MM.